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A. Palotie on työryhmän Int IBD Genetics Consortium jäsen. One goal of human genetics is to understand the genetic basis of disease, a challenge for diseases of complex inheritance because risk alleles are few relative to the vast set of benign variants. Risk variants are often sought by association studies in which allele frequencies in case subjects are contrasted with those from population-based samples used as control subjects. In an ideal world we would know population-level allele frequencies, releasing researchers to focus on case subjects. We argue this ideal is possible, at least theoretically, and we outline a path to achieving it in reality. If such a resource were to exist, it would yield ample savings and would facilitate the effective use of data repositories by removing administrative and technical barriers. We call this concept the Universal Control Repository Network (UNICORN), a means to perform association analyses without necessitating direct access to individual-level control data. Our approach to UNICORN uses existing genetic resources and various statistical tools to analyze these data, including hierarchical clustering with spectral analysis of ancestry; and empirical Bayesian analysis along with Gaussian spatial processes to estimate ancestry-specific allele frequencies. We demonstrate our approach using tens of thousands of control subjects from studies of Crohn disease, showing how it controls false positives, provides power similar to that achieved when all control data are directly accessible, and enhances power when control data are limiting or even imperfectly matched ancestrally. These results highlight how UNICORN can enable reliable, powerful, and convenient genetic association analyses without access to the individual-level data.

Background & Aims Biomarkers are needed for early detection of Crohn’s disease (CD) and ulcerative colitis (UC) or to predict patient outcomes. Glycosylation is a common and complex posttranslational modification of proteins that affects their structure and activity. We compared plasma N-glycosylation profiles between patients with CD or UC and healthy individuals (controls). Methods We analyzed the total plasma N-glycomes of 2635 patients with inflammatory bowel diseases and 996 controls by mass spectrometry with a linkage-specific sialic acid derivatization technique. Plasma samples were acquired from 2 hospitals in Italy (discovery cohort, 1989 patients with inflammatory bowel disease [IBD] and 570 controls) and 1 medical center in the United States (validation cohort, 646 cases of IBD and 426 controls). Sixty-three glycoforms met our criteria for relative quantification and were extracted from the raw data with the software MassyTools. Common features shared by the glycan compositions were combined in 78 derived traits, including the number of antennae of complex-type glycans and levels of fucosylation, bisection, galactosylation, and sialylation. Associations of plasma N-glycomes with age, sex, CD, UC, and IBD-related parameters such as disease location, surgery and medication, level of C-reactive protein, and sedimentation rate were tested by linear and logistic regression. Results Plasma samples from patients with IBD had a higher abundance of large-size glycans compared with controls, a decreased relative abundance of hybrid and high-mannose structures, lower fucosylation, lower galactosylation, and higher sialylation (α2,3- and α2,6-linked). We could discriminate plasma from patients with CD from that of patients with UC based on higher bisection, lower galactosylation, and higher sialylation (α2,3-linked). Glycosylation patterns were associated with disease location and progression, the need for a more potent medication, and surgery. These results were replicated in a large independent cohort. Conclusions We performed high-throughput analysis to compare total plasma N-glycomes of individuals with vs without IBD and to identify patterns associated with disease features and the need for treatment. These profiles might be used in diagnosis and for predicting patients’ responses to treatment.

In the past decade, there have been fundamental advances in our understanding of genetic factors that contribute to the inflammatory bowel diseases (IBDs) Crohn’s disease and ulcerative colitis. The latest international collaborative studies have brought the number of IBD susceptibility gene loci to 163. However, genetic factors account for only a portion of overall disease variance, indicating a need to better explore gene-environment interactions in the development of IBD. Epigenetic factors can mediate interactions between the environment and the genome; their study could provide new insight into the pathogenesis of IBD. We review recent progress in identification of genetic factors associated with IBD and discuss epigenetic mechanisms that could affect development and progression of IBD.

Genetic association studies have identified 210 risk loci for inflammatory bowel disease1–7, which have revealed fundamental aspects of the molecular biology of the disease, including the roles of autophagy and Th17 cell signaling and development. We performed a genome-wide association study of 25,305 individuals, and meta-analyzed with published summary statistics, yielding a total sample size of 59,957 subjects. We identified 26 new genome-wide significant loci, three of which contain integrin genes that encode molecules in pathways identified as important therapeutic targets in inflammatory bowel disease. The associated variants are also correlated with expression changes in response to immune stimulus at two of these genes (ITGA4, ITGB8) and at two previously implicated integrin loci (ITGAL, ICAM1). In all four cases, the stimulus-dependent expression increasing allele also increases disease risk. We applied summary statistic fine-mapping and identified likely causal missense variants in the primary immune deficiency genePLCG2and the negative regulator of inflammation,SLAMF8. Our results demonstrate that new common variant associations continue to identify genes and pathways of relevance to therapeutic target identification and prioritization.

IntroductionThere is an unmet need for novel blood based biomarkers that offer timely and accurate diagnostic and prognostic testing in Inflammatory Bowel Diseases (IBD). We aimed to investigate the diagnostic and prognostic utility of serum calprotectin (SC) in IBD.MethodsA total of 171 patients (n=96 IBD, n=75 non-IBD) were prospectively recruited. A multi biomarker model was derived using multivariable logistic regression analysis. Cox proportional hazards model was derived to assess the contribution of each variable to disease outcomes.ResultsSC correlated strongly with current biomarkers including faecal calprotectin (FC) (n=50, rho= 0.50, p=1.6x10-437 ). SC was the strongest individual predictor of IBD diagnosis (odds ratio (OR): 9.37(95%CI: 2.82-34.68), p=4.00×10-438 ) compared with other markers (CRP: OR 8.52(95%CI: 2.75-28.63), p=2.80×10-439 ); albumin: OR 6.12(95%CI: 1.82-22.16), p=0.004). In a subset of 50 patients with paired SC and FC, the area under receiver operating characteristic discriminating IBD from controls was better for FC than SC (0.99, (95% CI 0.87-1.00) and 0.87 (95% CI:0.78-0.97) respectively; p=0.01).43 At follow up (median 342 days; IQR: 88-563), SC predicted treatment escalation and/or44 surgery in IBD (HR 2.7, 95% CI: 1.1-4.9), in particular CD (HR 4.2, 95% CI 1.2-15.3).Page 2 of 73ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901American Journal of GastroenterologyFor Peer Review3A model incorporating SC and either CRP or albumin has a positive likelihood 45 ratio of 24.1446 for IBD. At 1 year, our prognostic model can predict treatment escalation in IBD in 65% of47 cases (95% CI: 43-79%) and 80% (95% CI: 31-94%) in CD if 2 or more blood marker48 criteria are met.49 Conclusions50 A diagnostic and prognostic model that combines SC and other blood-based biomarkers51 accurately predicts the inflammatory burden in IBD and has the potential to predict disease52 and its outcomes. Our data warrants further detailed exploration and validation in large multi53centre cohorts.

Epigenetic alterations may provide important insights into gene-environment interaction in inflammatory bowel disease (IBD). Here we observe epigenome-wide DNA methylation differences in 240 newly-diagnosed IBD cases and 190 controls. These include 439 differentially methylated positions (DMPs) and 5 differentially methylated regions (DMRs), which we study in detail using whole genome bisulphite sequencing. We replicate the top DMP (RPS6KA2) and DMRs (VMP1, ITGB2 and TXK) in an independent cohort. Using paired genetic and epigenetic data, we delineate methylation quantitative trait loci; VMP1/microRNA-21 methylation associates with two polymorphisms in linkage disequilibrium with a known IBD susceptibility variant. Separated cell data shows that IBD-associated hypermethylation within the TXK promoter region negatively correlates with gene expression in whole-blood and CD8+ T cells, but not other cell types. Thus, site-specific DNA methylation changes in IBD relate to underlying genotype and associate with cell-specific alteration in gene expression., Epigenetic perturbations may be an important factor in diseases where both genes and environment play a role. Here, Ventham and colleagues show that DNA methylation changes in inflammatory bowel disease are related to the underlying genotype, and are associated with cell-specific changes to gene expression.

Background: Inflammatory bowel diseases (IBDs) are a result of interactions between luminal pathogens and the intestinal immune response. Cyclooxygenase-2 (COX-2) plays a key role in the regulation of the inflammatory response upon stimulation by luminal pathogens via Toll-like receptors. Methods: Genotypes of the COX-2/PTGS2/PGHS2 A-1195G (rs689466), G-765C (rs20417), and T8473C (rs5275) polymorphisms were assessed in a Scottish and Danish case–control study including 732 Crohn's disease (CD) cases, 973 ulcerative colitis (UC) cases, and 1157 healthy controls using logistic regression. Results: Carriers of the COX-2 A-1195G variant allele had increased risk of UC (odds ratio [OR], 95% confidence interval [CI] = 1.25 [1.02–1.54], P = 0.03) and of both UC and IBD among never smokers (OR [95% CI] = 1.47 [1.11–1.96], P = 0.01 and OR [95% CI] = 1.37 [1.06–1.77], P = 0.02, respectively). Furthermore, this variant genotype was associated with increased risk of diagnosis of UC before age 40 years and with extensive UC (OR [95% CI] = 1.34 [1.11–1.62], P = 0.002 and OR [95% CI] = 1.32 [1.03–1.69], P = 0.03, respectively). Conclusions: COX-2 A-1195G polymorphism was associated with the risk of UC, especially among never-smokers, suggesting that low activity of COX-2 may predispose to UC. Our results suggest that inclusion of smoking status may be essential for the evaluation of the role of genetic predisposition to IBD. (Inflamm Bowel Dis 2011)

Autophagy is a cellular pathway involved in protein and organelle degradation, which is likely to represent an innate adaptation to starvation. In times of nutrient deficiency, the cell can self-digest and recycle some nonessential components through nonselective autophagy, thus sustaining minimal growth requirements until a food source becomes available. Over recent years, autophagy has been implicated in an increasing number of clinical scenarios, notably infectious diseases, cancer, neurodegenerative diseases, and autoimmunity. The recent identification of the importance of autophagy genes in the genetic susceptibility to Crohn's disease suggests that a selective autophagic response may play a crucial role in the pathogenesis of common complex immune-mediated diseases. In this review, we discuss the autophagic mechanisms, their molecular regulation, and summarize their clinical relevance. This progress has led to great interest in the therapeutic potential of manipulation of both selective and nonselective autophagy in established disease.

The high incidence of Scottish Crohn's disease (CD) is not explained by the common three NOD2/CARD15 variants. We aimed to identify population-specific NOD2/CARD15 coding variants. A total of 1478 (320 inflammatory bowel disease patients

The rs2241880A/G variant of the ATG16L1 gene has been associated with susceptibility to ileal Crohn's disease (CD) in adults. Our aim was to assess whether germline variation of ATG16L1 acts as an independent determinant of susceptibility to childhood-onset CD in the high-incidence Scottish population.In all, 2195 subjects (361 children (inflammatory bowel disease [IBD] diagnosis17 years), their parents (n = 634), 855 adult IBD patients, and 345 controls were genotyped. Case-control analysis was powered to detect effect sizes with an odds ratio (OR)1.39 in pediatric CD. Case-control analysis, transmission disequilibrium testing (TDT), analysis of variance (ANOVA) of growth parameter z-scores, Kruskal-Wallis test (age at diagnosis), and multifactorial genotype-phenotype analysis (Montreal classification) were performed. 7.8% of pediatric CD patients and 37.2% of adult CD patients had pure ileal disease.We confirmed the association of the rs2241880G-allele with adult-onset CD (60.7% versus controls 53.9%, P = 0.01, OR 1.32, 95% confidence interval [CI] 1.07-1.63) in contrast to childhood-onset CD (54.1% versus controls, P = 0.95, OR 1.01, 95% CI 0.80-1.26). TDT analysis was negative. Genotype-phenotype analysis demonstrated an association of pure ileal disease with the rs2241880G-allele (P = 0.02, OR 1.34, 95% CI 1.03-1.74). Using binary logistic regression analysis we confirmed the effect of rs2241880 genotype (GG) on ileal disease versus colonic disease (P = 0.03, OR 2.43, 95% CI 1.05-5.65). ATG16L1 genotype did not influence age at CD diagnosis. ANOVA of z-scores of height, weight, and body mass index (BMI) at CD diagnosis in children showed no association with genotype.The ATG16L1 variant is associated with susceptibility to adult CD in Scotland, but not early-onset disease. These contrasting effects are primarily driven by differences in disease location between early-onset and adult-onset disease.

SUMMARY Background Osteoporosis is a recognized complication of inflammatory bowel disease (IBD). Aim To investigate the role of environmental factors and vitamin D receptor (VDR) variants on the prevalence of osteoporosis. Methods DEXA scans and case note review were performed on 440 IBD patients from 1997 to 2006. All the IBD patients and 240 healthy controls were genotyped for VDR variants Taq-1 and Apa-1 using PCR-RFLP. Results Osteoporosis and osteopenia rates were 15% and 18% for IBD, 16% and 18% for Crohn’s disease (CD) and 13% and 19% for ulcerative colitis, respectively. On univariate analysis of the CD patients, low body mass index (BMI

Objective To assess the contribution of the 113 G→A missense mutation within the discs, large homolog 5 (DLG5) gene in childhood-onset inflammatory bowel disease (IBD) in Scotland. Study design Two-hundred and ninety-six children with IBD were studied. Parental DNA was also collected for transmission disequilibrium testing (TDT) analysis. Genotyping was performed by TaqMan®. Genotype-phenotype analysis was also undertaken. Socioeconomic status was assigned using a deprivation category (DepCat) score 1 through 7 (1 = most affluent). Results TDT analysis demonstrated a significant association with IBD ( P = .045). On unifactorial analysis, 113A carriage was associated with: (1) higher social class (DepCat 1 compared with 2-7, and 1-2 compared with 3-7) (66.7% vs 22.6%, P = .0005, OR 6.84 [1.99-23.55] and 37.2% vs 22.2%, P = .03, OR 2.08 [1.04-4.17], respectively); (2) higher height centile (>75 th centile vs th centile) (42.9% vs 23.1%, P = .01, OR 2.50 [1.18-5.28]); and (3) male sex in Crohn's disease (CD) (29.3% vs 16.9%, P = .04, OR 2.04 [1.01-4.11]). Multifactorial analysis demonstrated that higher social class (DepCat 1) was independently associated with carriage of variants of 113A ( P = .001, OR=6.92 [2.24-21.33]). Conclusions DLG5 113A is associated with increased susceptibility to IBD in Scottish children. The effect may be most marked for those children living in relative affluence.

The discovery of nucleotide-binding oligomerization domain 2/caspase recruitment domain-containing protein 15 (NOD2/CARD15) as the first susceptibility gene in Crohn's disease (CD) has shifted the focus of research into the pathogenesis of inflammatory bowel disease (IBD) firmly to the innate immune response and the integrity of the epithelial barrier. The subsequent implication in IBD of variant alleles of OCTN, DLG5, MDR1, and TLRs has provided further support for a new, more complex model of innate immunity function in the gastrointestinal tract. In this review, we examine the recent advances in our understanding of the influence of genetics of the innate immune response on IBD. We will focus on germline variation of genes encoding pathogen-recognition receptors, proteins involved in epithelial homeostasis and secreted antimicrobial proteins. Copyright © 2006 Crohn's & Colitis Foundation of America, Inc.

Recent data have controversially suggested that variants of the organic cation transport genes SLC22A4 (OCTN1) and SLC22A5 (OCTN2) are responsible for the contribution of IBD5 to disease susceptibility in Crohn's disease (CD). The objective of this study was to assess the contribution of the SLC22A4 variant (1672T) and SLC22A5 variant (-207C) together with three IBD5 haplotype markers in the previously uninvestigated Swedish CD population.The study comprised 178 CD patients and 143 healthy controls (HC). Genotyping for IBD5 single nucleotide polymorphisms (SNPs) IGR2096a_1, IGR2198a_1, IGR2230a_1, SLC22A4 1672T and SLC22A5 -207C was carried out using the TaqMan system. Associations with disease susceptibility and disease phenotype were investigated.Strong linkage disequilibrium was observed between the investigated SNPs (D prime0.92). IGR2096a_1 allelic frequency and homozygosity rates were associated with CD (44% CD versus 33.8% HC, p=0.008, OR=1.55 and 20% CD versus 12% HC, p=0.04, OR=1.93, respectively). Variant allelic frequency of SLC22A4, 1672T (44% versus 36%, p=0.03, OR=1.4) and homozygosity for the SLC22A4, SLC22A5 TC haplotype (1672T, -207C) (21.3% versus 12%, p=0.03, OR=1.78, population attributable risk (PAR)=11%) were associated with CD. There was no association between the allelic frequency of SLC22A5 and CD (46.6% CD versus 41.5% HC, p=0.82). The association of the TC haplotype with CD was not independent of the SNPs representing the extended IBD5 linkage interval.The IBD5 locus is associated with CD in the Swedish population. The strongest association is with the marker SNP IGR2096a_1, lying p-telomeric to SLC22A4 and SLC22A5. The effect of the TC haplotype was not an independent determinant in this population.

Several lines of evidence suggest a role for the multidrug resistance gene (ABCB1/MDR1) and its product, P-glycoprotein 170, in the pathogenesis of inflammatory bowel disease (IBD). In addition, P-glycoprotein activity determines bioavailability of many drugs used regularly in many medical specialities, and ABCB/ MDR1 variation appears to be a critical pharmacogenetic determinant. We have utilized a gene-wide haplotype tagging approach to further define the identity of germ-line variations in the ABCB1/MDR1 gene contributing to IBD susceptibility. Six haplotype tagging single nucleotide polymorphisms (tSNPs) representing the haplotypic variations of the ABCB1/MDR1 gene were identified initially following the characterization of the haplotype structure of this gene in 24 Centre d'Etude du Polymorphisme Humain Caucasian trios. Genotyping was performed in 249 ulcerative colitis (UC) and 179 Crohn's disease (CD) patients and 260 healthy controls. Using log-likelihood analysis, we observed a highly significant association between the common haplotypes and UC (P = 4.22 x 10 -7 ) but not CD (P = 0.22). This significant association was critically dependent on one tSNP, intronic variant rs3789243. All haplotypes with this variant retained a highly significant association (P = 3.2 x 10 -7 -3.6 x 10 -12 ), whereas significance was lost when rs3789243 was dropped in systematic haplotypic analysis. The effect of this tSNP was independent of C3435T SNP, previously suggested to be the critical variant in disease susceptibility and drug transport. The association with UC was shown to be strongest with the phenotype of extensive disease (P = 1.7 x 10 -7 ). This 'candidate gene' approach provides compelling evidence to support the contribution of the ABCB1/MDR1 gene in determining risk to UC but not to CD and provides new insights into the localization of the critical susceptibility determinants within the gene. In addition, these findings have potentially important implications in the application of pharmacogenetics across a range of common diseases, including HIV, epilepsy and colorectal cancer.

Background & Aims: Recent data suggest that polymorphisms in the organic cation transporter (OCTN) genes OCTN1 (SLC22A4) and OCTN2 (SLC22A5) represent disease-causing mutations within the IBD5 locus (chromosome 5q31). We investigated associations with disease susceptibility, phenotype, and evidence for epistasis with CARD15 in 679 patients with Crohn’s disease (CD) or ulcerative colitis (UC). Methods: A total of 374 patients with CD, 305 patients with UC, and 294 healthy controls (HCs) were studied. Genotyping for single nucleotide polymorphisms IGR2096, IGR2198, and IGR2230, OCTN1 variant (SLC22A4 1672C→T), and OCTN2 variant (SLC22A5 −207G→C) was performed using the TaqMan system. Results: The IBD5 OCTN1 and OCTN2 polymorphisms were in strong linkage disequilibrium (D′, >0.959). IGR2198 variant allele frequency (49.1% vs 40.8%; P = .0046) and homozygosity (21% vs 14.8%; P = .044) were associated with CD versus HCs. Variant allelic frequency of OCTN1 (53.6% vs 43%; P = .0008) and OCTN2 (56.1% vs 48.4%; P = .0092) polymorphisms and homozygosity for the OCTN1/2-TC haplotype (28.4% vs 16%; P = .0042) were associated with CD versus HCs. IGR2198 homozygosity and TC homozygosity were associated with stricturing/penetrating disease at follow-up (P = .011 and P = .011, respectively) and disease progression (P = .038 and P = .049, respectively) on univariate analysis and with need for surgery on multivariate analysis (P = .016 and P = .004, respectively). In the absence of the IBD5 risk haplotype, no association of OCTN1/2 variants with CD was detected. No associations were seen with UC. Conclusions: The IBD5 locus influences susceptibility, progression, and need for surgery in CD. However, the contribution of OCTN1/2 variants is not independent of the IBD5 haplotype; a causative role for these genes remains plausible but is not yet proven. Further genetic, functional, and expression data are now required.

Background & Aims: The MDR1 gene encodes P-glycoprotein 170, an efflux transporter that is highly expressed in intestinal epithelial cells. The MDR1 exonic single nucleotide polymorphisms (SNPs) C3435T and G2677T have been shown to correlate with activity/expression of P-glycoprotein 170. Methods: This was a case-control analysis of MDR1 C3435T and G2677T SNPs in a large well-characterized Scottish white cohort (335 with ulcerative colitis [UC], 268 with Crohn's disease [CD], and 370 healthy controls). We conducted 2-locus haplotype and detailed univariate and multivariate genotypic-phenotypic analyses. Results: The MDR1 3435 TT genotype (34.6% vs 26.5%; P = .04; odds ratio [OR], 1.60; 95% confidence interval [95% CI], 1.04–2.44) and T-allelic frequencies (58.2% vs 52.8%; P = .02; OR, 1.28; 95% CI, 1.03–1.58) were significantly higher in patients with UC compared with controls. No association was seen with CD. The association was strongest with extensive UC (TT genotype: 42.4% vs 26.5%; P = .003; OR, 2.64; 95% CI, 1.34–4.99; and T allele: 63.9% vs 52.8%; P = .009; OR, 1.70; 95% CI, 1.24–2.29), and this was also confirmed on multivariate analysis ( P = .007). The G2677T SNP was not associated with UC or CD. These 2 SNPs lie in linkage disequilibrium in our population (D′, .8–.9; r 2 , .7–.8). Two-locus haplotypes showed both positive (3435T/G2677 haplotype: P = .03; OR, 1.44) and negative (C3435/2677T haplotype: P = .002; OR, .35) associations with UC. Homozygotes for the haplotype 3435T/G2677 were significantly increased in UC ( P = .017; OR, 8.88; 95% CI, 1.10–71.45). Conclusions: Allelic variations of the MDR1 gene determine disease extent as well as susceptibility to UC in the Scottish population. The present data strongly implicate the C3435T SNP, although the 2-locus haplotype data underline the need for further detailed haplotypic studies.

Progress in the genetics of complex diseases has been slow over the past two decades compared to many simple Mendelian traits. However, rapid advances are now being made in inflammatory bowel disease genetics, leading already to identification of the first gene linked to Crohn's disease susceptibility: NOD2/CARD15. Since its discovery three years ago, there has been replication of the association of NOD2/CARD15 mutations with Crohn's disease in many populations, together with identification of phenotypic correlations. Functional studies promise to increase understanding of the primary pathophysiology involved in Crohn's disease and these discoveries may yet change clinical practice.

SUMMARY Anti-Saccharomyces cerevisiae antibodies (ASCAs) have been proposed as serological markers, which may differentiate Crohn's disease (CD) from ulcerative colitis (UC) and predict disease phenotype. Their importance in pathogenesis is unproven. We investigated the relationship between ASCAs, disease phenotype and NOD2/CARD15 genotype in CD and whether ASCAs were related to antibodies to other fungal proteins. Serum from 228 patients [143 CD, 75 UC, 10 with indeterminate colitis (IC)] and 78 healthy controls (HC) were assayed for ASCA. Antibodies (IgA, IgG) to other fungal proteins (Fusarium species ATC20334, Mycoprotein) were measured in the same samples using an in-house enzyme-linked immunosorbent assay (ELISA) assay. ASCAs were present in 57% of CD, 19% of UC, 30% of IC and 8% of HCs. ASCA-positive status was a predictor for CD with sensitivity of 57%, specificity of 87%, positive predictive value of 78% and negative predictive value of 68%. ASCA was associated with proximal (gastroduodenal and small bowel involvement) rather than purely colonic disease (P

Background Exploring DNA methylation in inflammatory bowel disease might provide an insight into the complex gene–environment interactions in disease pathogenesis. Our study aimed to characterise disease-associated methylation changes in newly diagnosed inflammatory bowel disease and explore its association with germline variation and gene expression. Methods Samples were obtained from new onset inflammatory bowel disease in six European centres (IBD-Character project). Genome-wide methylation was measured (450k platform, Illumina, San Diego, CA, USA) in 641 whole blood DNA samples (298 controls, 150 Crohn's disease, 167 ulcerative colitis, 26 inflammatory bowel disease unclassified). Genotyping and gene expression were performed using HumanOmniExpressExome-8 BeadChips (Illumina) and Ion AmpliSeq (ThermoFisher, Waltham, MA, USA) platforms, respectively. Correlation and pathway analyses were performed between the top differentially methylated regions and gene expression. Findings M195 probes exhibited Holm-significant inflammatory bowel disease-associated methylation differences, including MIR21 (p=3·7 × 10 − 20 ) and RPS6KA2 (p=1·1 × 10 −19 ) with only one probe differentiating Crohn's disease from ulcerative colitis ( NAV2 , p=6·82 × 10 −8 ). Paired genetic and methylation data showed 1037 significant methylation quantitative trait loci indicating a genetic influence on several key loci: RPS6KA2 (p=8·6 × 10 −34 ) and MIR21 (rs8078424, p=4·4 × 10 − 25 ; rs10853015, p=7·4 × 10 −21 ). 1543 differentially methylated regions (DMRs) were identified and included MIR21 , RPS6KA2 , and TNF . These DMRs mapped to 8214 mRNA profiles of which 1916 showed Holm-significant correlation with methylation such as IL32 (seven probes: r =–0·50 to −0·66, p=6·0 × 10 −76 ). GO term analyses of these highly correlated genes revealed pathways that regulate cell–cell adhesion and immune cell differentiation. Interpretation Our data provide a comprehensive multicentre genome-wide profile of the circulating methylome and give an insight into the complex interplay between differential methylation, germline variation, and gene expression in immune mediated disease. Funding RK and AA are funded by IBD-Character (a European Union 7th Framework Programme project).

DNA double strand break (DSB) repair by non‐homologous end joining (NHEJ) in mammalian cells requires the Ku70–Ku80 heterodimer, the DNA‐PK catalytic subunit DNA‐PKcs, as well as DNA ligase IV and Xrcc4. NHEJ of plasmid DSBs in Saccharomyces cerevisiae requires Ku, Xrcc4 and DNA ligase IV, as well as Mre11, Rad50, Xrs2 and DNA damage checkpoint proteins. Saccharomyces cerevisiae Ku is also required for telomere length maintenance and transcriptional silencing. We have characterized NHEJ in Schizosaccharomyces pombe using an extrachromosomal assay and find that, as anticipated, it is Ku70 and DNA ligase IV dependent. Unexpectedly, we find that Rad32, Rad50 (the S.pombe homologues of Mre11 and Rad50, respectively) and checkpoint proteins are not required for NHEJ. Furthermore, although S.pombe Ku70 is required for maintenance of telomere length, it is dispensable for transcriptional silencing at telomeres and is located throughout the nucleus rather than concentrated at the telomeres. Together, these results provide insight into the mechanism of NHEJ and contrast significantly with recent studies in S.cerevisiae .

A genome- wide association scan in individuals with Crohn's disease by the Wellcome Trust Case Control Consortium detected strong association at four novel loci. We tested 37 SNPs from these and other loci for association in an independent case- control sample. We obtained replication for the autophagy- inducing IRGM gene on chromosome 5q33.1 ( replication P = 6.6 x 10(-4), combined P = 2.1 x 10(-10)) and for nine other loci, including NKX2- 3, PTPN2 and gene deserts on chromosomes 1q and 5p13.

During meiotic prophase, chromosomes frequently adopt a bouquet-like arrangement, with their telomeres clustered close to the nuclear periphery. A dramatic example of this occurs in the fission yeast, Schizosaccharomyces pombe, where all telomeres aggregate adjacent to the spindle pole body (SPB). Nuclei then undergo rapid traverses of the cell, known as 'horsetail' movement, which is led by the SPB dragging telomeres and chromosomes behind. This process may initiate or facilitate chromosome pairing before recombination and meiosis. With the aim of identifying components involved in telomere structure and function, we report here the isolation of S. pombe mutants defective in the ability to impose transcriptional silencing on genes placed near telomeres. Two of these mutants, lot2-s17 and lot3-uv3, also display a dramatic lengthening of telomeric repeats. lot3-uv3 carries a mutation in Taz1, a telomere-binding protein containing a Myb-like motif similar to two human telomere-binding proteins. Meiosis is aberrant in these mutant yeast strains, and our analysis demonstrates a decreased association of telomeres with the SPB in meiotic prophase. This results in defective 'horsetail' movement, a significant reduction in recombination, low spore viability and chromosome missegregation through meiosis.

Telomeres, the specialized nucleoprotein structures that comprise the ends of eukaryotic chromosomes, are essential for complete replication, and regulation of their length has been a focus of research on tumorigenesis. In the budding yeast Saccharomyces cerevisiae, the protein Rap1p binds to telomeric DNA and functions in the regulation of telomere length. A human telomere protein, hTRF (human TTAGGG repeat factor) binds the telomere sequence in vitro and localizes to telomeres cytologically, but its functions are not yet known. Here we use a genetic screen to identify a telomere protein in fission yeast, Taz1p (telomere-associated in Schizosaccharomyces pombe), that shares homology to the Myb proto-oncogene DNA-binding domain with hTRF. Disruption or deletion of the taz1+ gene causes a massive increase in telomere length. Taz1p is required for the repression of telomere-adjacent gene expression and for normal meiosis or sporulation. It may be a negative regulator of the telomere-replicating enzyme, telomerase, or may protect against activation of telomerase-independent pathways of telomere elongation.

Introduction There is an unmet need for novel blood based biomarkers that offer timely and accurate diagnostic and prognostic testing in Inflammatory Bowel Diseases (IBD). We aimed to investigate the diagnostic and prognostic utility of serum calprotectin (SC) in IBD Methods A total of 156 patients (82 IBD and 74 non-IBD) were sampled within 90 days from diagnosis (median 0 days; IQR 0–7). A multibiomarker diagnostic and prognostic model was derived using multivariable logistic regression analysis. Treatment escalation was defined as the need for escalation and establishment of 2 or more immunomodulatory therapies and/or surgery for disease flare after initial induction of disease remission (criteria previously used by Lee et al) (1). Cox proportional hazards model was derived to assess the contribution of each variable to disease outcomes Results SC correlated strongly with current biomarkers including CRP (rho = 0.60, p = 1.4x10−16) faecal calprotectin (FC) (rho = 0.51, p = 1.6x10−4). Paired FC was available within 30 days (median 0 days, IQR: -4 to5 days) of SC in 50 patients (IBD n = 30, non-IBD n = 20).The area under receiver operating characteristic discriminating IBD from controls was similar for FC and SC(0.95, 95% CI 0.87–1.00 and 0.89, 95%CI 0.81–0.98 respectively;p = 0.36). SC was the strongest individual predictor of IBD diagnosis (odds ratio (OR): 12.33 (95% CI 4.48–38.33, p = 3.5×10−6) compared with other markers (CRP: OR 4.44, CI 1.58–12.90; albumin: OR 5.65, CI 1.98–17.16). At follow up (median 342 days; IQR: 88–563),a total of 1 (2%), 16 (47%),23 (51%) patients required treatment escalation in the IBDU, CD and UC group respectively.SC predicted treatment escalation and/or surgery in IBD (HR 2.4, 95% CI: 1.1–4.9), in particular CD (HR 4.1, 95% CI 1.1–14.7). A model incorporating SC, CRP and albumin has a positive likelihood ratio of 20.03 for IBD.At 1 year, our prognostic model can predict treatment escalation in IBD in 65% of cases (95% CI: 43–79%) and 80% (95% CI: 31–94%) in CD if 2 or more blood marker criteria are met. Conclusion Sampling faeces can be a hurdle for patients and some individuals can decline FC testing. These factors impact on the practical utility of FC.SC shows promise as a diagnostic and prognostic biomarker in IBD. Our findings warrant further exploration and validation within large multicentre cohorts. Reference 1 Lee JC, et al. Gene expression profiling of CD8+ T cells predicts prognosis in patients with Crohn disease and ulcerative colitis. J Clin Invest 2011;121:4170–9. Disclosure of Interest None Declared

Proximity extension assay immunoassay technology identifies novel serum biomarkers that can diagnose and classify inflammatory bowel diseases : IBD Character Consortium

International audience; The ura4+ gene displays phenotypes consistent with variegated expression when inserted at 11 sites throughout fission yeast centromere 1. An abrupt transition occurs between the zone of centromeric repression and two adjacent expressed sites. Mutations in six genes alleviate repression of the silent-mating type loci and of ura4+ expressed from a site adjacent to the silent locus, mat3-M. Defects at all six loci affect repression of the ura4+ gene adjacent to telomeres and at the three centromeric sites tested. The clr4-S5 and rik1-304 mutations cause the most dramatic derepression at two out of three sites within cen1. All six mutations had only slight or intermediate effects on a third site in the center of cen1 or on telomeric repression. Strains with lesions at the clr4, rik1, and swi6 loci have highly elevated rates of chromosome loss. We propose that the products of these genes are integral in the assembly of a heterochromatin-like structure, with distinct domains, enclosing the entire centromeric region that reduces or excludes access to transcription factors. The formation of this heterochromatic structure may be an absolute requirement for the formation of a fully functional centromere.

In recent years the inflammatory bowel diseases (IBD)—Crohn's disease and ulcerative colitis—have emerged as a model for all complex traits in the successful dissection of the molecular basis of inherited susceptibility by non-parametric linkage analysis1 2 and genome-wide association studies (GWAS).3–5 The recent meta-analyses of GWAS carried out by the International Inflammatory Bowel Disease Genetics Consortium have now reported the identification of 99 susceptibility loci involved in the pathogenesis of Crohn's disease and ulcerative colitis.4 5 These successes in candidate gene discovery have provided new insights into the pathogenetic mechanisms involved in the chronic intestinal inflammation associated with IBD. These data provide strong evidence that Crohn's disease and ulcerative colitis are related complex diseases, sharing some, but not all, susceptibility loci.5 It is clear that while some pathogenic pathways are implicated in both Crohn's disease and ulcerative colitis—notably, interleukin (IL)-23 signalling6 and Th17 cell activation,7 other innate immune mechanisms appear to be implicated in Crohn's disease only (eg, autophagy, and NOD2 signalling). Similarly, aspects of the maintenance of epithelial barrier function are most strongly implicated in ulcerative colitis.8 Furthermore, it is clear that many of the genetic determinants involved in IBD are also implicated in other complex disorders, such as type I diabetes and coeliac disease ( in press ).9 Both expected and unexpected associations are evident, and have provided avenues for further research. Along with this exciting progress, however, new challenges have emerged for researchers in this complex field. Of the estimated heritability of IBD, which has been derived from concordance rates in twin pairs

Background: Germline variation in the 71 Crohn's disease (CD) loci implicated by genome-wide association studies (GWAS) only accounts for approximately 25% of estimated heritability. The contribution of epigenetic alterations to disease pathogenesis is emerging as a research priority. Materials and Methods: The methylation status of 27,578 CpG sites across the genome was analyzed using the Illumina Human Methylation27 assay in DNA extracted from whole blood samples from 40 adult females (21 ileal CD, 19 healthy controls) and 16 girls with childhood-onset CD, all nonsmokers. Our primary analysis compared methylation profiles in adult cases and controls. Results: Our data define a global methylation profile characteristic of ileal CD. In all, 1117 sites were differentially methylated (corrected P < 0.01); 50 showed significantly altered methylation in cases compared with controls (uncorrected P < 10−6, corrected P < 0.0006), including genes altering immune activation: MAPK13, FASLG, PRF1, S100A13, RIPK3, and IL-21R. Gene ontology analyses implicated immunity-related pathways as targets of epigenetic modification (immune system processes [P = 1.3 × 10−22], immune response [P = 8.1 × 10−16], defense responses to bacteria [P = 1.8 × 10−15]). Ingenuity canonical pathway analyses implicated dendritic cell activity (P = 2.4 × 10−8) and differential regulation of cytokines by interleukin (IL)-17A and IL-17F (P = 5.8 × 10−7). We identified a significant enrichment of methylation changes within 50 kb of CD GWAS loci (8.6-fold [P = 0.021] in adults; 2.4-fold [P = 0.009] in adults and children combined), including IL-27, IL-19, TNF, MST1, and NOD2. Methylation status was predictive of disease status (sensitivity 0.71, specificity 0.83). Disease activity, drug therapy, NOD2 and DNMT3A genotypes were not associated with methylation changes. Conclusions: These data provide an important insight into the impact of epigenetic mechanisms in the pathogenesis of CD. (Inflamm Bowel Dis 2011;)

Background: Pediatric inflammatory bowel disease (IBD) has a high prevalence of coexistent atopy. Filaggrin (FLG) loss-of-function variants (null-alleles) are associated with eczema and asthma in association with eczema. The aim was to assess the contribution of FLG null-alleles to pediatric IBD susceptibility and to coexistent atopy (eczema, asthma, allergic rhinitis, or food allergy). Methods: FLG variants (R501X and 2282del4) were genotyped in 403 children with IBD, 683 parents, and 996 population controls. Results: In all, 11% of IBD patients carried at least 1 FLG null-allele compared to 11% of population controls (P > 0.4). Carriage of 1 or more null-alleles in patients with atopy (present in 52% of IBD patients) differed from IBD patients without atopy (14% versus 6%, P = 0.01; odds ratio [OR] 2.4, 95% confidence interval [CI] 1.2-5.1). The effect of FLG null-alleles was strongest for eczema (19% versus 7%, P = 0.0003; OR 3.3, 95% CI 1.7-6.6) and food allergy (28% versus 8%, P = 0.0001; OR 4.5, 95% CI 2.0-10.0). The presence of more than 1 atopic disease tended to increase the associated OR: eczema + asthma (23% versus 7%, P = 0.001; OR 3.9, 95% CI 1.6-9.1), eczema + asthma + allergic rhinitis (29% versus 7%, P = 0.0006; OR 5.4, 95% CI 1.9-15.4) and eczema + asthma + allergic rhinitis + food allergy (45% versus 6%, P < 10-4; OR 12.2, 95% CI 3.2-46.3). Logistic regression analysis of IBD cases confirmed the association of carriage of an FLG null-allele with atopy (P = 0.01; OR 2.4, 95% CI 1.2-5.1) and co-occurrence of different forms of atopy (P = 0.003; OR 3.5, 95% CI 1.5-8.1). Conclusions: Filaggrin null-alleles have no effect on IBD susceptibility but contribute to coexistent eczema and food allergy. Copyright © 2009 Crohn's & Colitis Foundation of America, Inc.

Background & Aims: Childhood-onset inflammatory bowel disease (IBD) might be etiologically different from adult-onset IBD. We analyzed disease phenotypes and progression of childhood-onset disease and compared them with characteristics of adult-onset disease in patients in Scotland. Methods: Anatomic locations and behaviors were assessed in 416 patients with childhood-onset (276 Crohn's disease [CD], 99 ulcerative colitis [UC], 41 IBD type unclassified [IBDU] diagnosed before seventeenth birthday) and 1297 patients with adult-onset (596 CD, 701 UC) IBD using the Montreal classification. Results: At the time of diagnosis in children, CD involved small bowel and colon (L3) in 51% (138/273), colon (L2) in 36%, and ileum (M) in 6%; the upper gastrointestinal (GI) tract (L4) was also affected in 51%. In 39%, the anatomic extent increased within 2 years. Behavioral characteristics progressed; 24% of children developed stricturing or penetrating complications within 4 years (vs 9% at diagnosis; P < .0001; odds ratio [OR], 3.32; 95% confidence interval [CI], 1.86 - 5.92). Compared with adults, childhood-onset disease was characterized by a "panenteric" phenotype (ileocolonic plus upper GI [L3 + L4]; 43% vs 3%; P

We report results of a nonsynonymous SNP scan for ulcerative colitis and identify a previously unknown susceptibility locus at ECM1. We also show that several risk loci are common to ulcerative colitis and Crohn's disease (IL23R, IL12B, HLA, NKX2-3 and MST1), whereas autophagy genes ATG16L1 and IRGM, along with NOD2 (also known as CARD15), are specific for Crohn's disease. These data provide the first detailed illustration of the genetic relationship between these common inflammatory bowel diseases.

Great progress in the understanding of the molecular genetics of inflammatory bowel disease (IBD) has been made over the last 10 years. Strong epidemiological evidence, based initially on concordance data in twin/family studies, led to the application of genome-wide linkage analysis involving multiply affected families and the identification of a number of susceptibility loci. Further characterization of the IBD1 locus on chromosome 16 led to the discovery of the NOD2/CARD15 gene as the first susceptibility gene in Crohn's disease for 2001. This landmark finding has led to a redirection of basic research in IBD with interest focused principally on regulation of the innate immune response and mucosal barrier function. Within the last year, the use of genome-wide association studies has provided new insights into primary pathogenetic mechanisms; several new genes such as the Interleukin-23 receptor (IL23R) and ATG16L1 (autophagy-related 16-like 1) genes are strongly implicated. Overall, these studies promise to change our fundamental understanding of IBD pathophysiology and to have implications for clinical practice.

BACKGROUND: DLG5 p.R30Q has been reported to be associated with Crohn disease (CD), but this association has not been replicated in most studies. A recent analysis of gender-stratified data from two case-control studies and two population cohorts found an association of DLG5 30Q with increased risk of CD in men but not in women and found differences between 30Q population frequencies for males and females. Male-female differences in population allele frequencies and male-specific risk could explain the difficulty in replicating the association with CD. METHODS: DLG5 R30Q genotype data were collected for patients with CD and controls from 11 studies that did not include gender-stratified allele counts in their published reports and tested for male-female frequency differences in controls and for case-control frequency differences in men and in women. RESULTS: The data showed no male-female allele frequency differences in controls. An exact conditional test gave marginal evidence that 30Q is associated with decreased risk of CD in women (p = 0.049, OR = 0.87, 95% CI 0.77 to 1.00). There was also a trend towards reduced 30Q frequencies in male patients with CD compared with male controls, but this was not significant at the 0.05 level (p = 0.058, OR = 0.87, 95% CI 0.74 to 1.01). When data from this study were combined with previously published, gender-stratified data, the 30Q allele was found to be associated with decreased risk of CD in women (p = 0.010, OR = 0.86, 95% CI 0.76 to 0.97), but not in men. CONCLUSION: DLG5 30Q is associated with a small reduction in risk of CD in women.

Background: Inflammatory bowel disease (IBD) is an epithelial barrier disease that is thought to result from a dysregulated interaction with bacteria in the intestine of genetically predisposed individuals. The cystic fibrosis transmembrane conductance regulator (CFTR), which is mutated in the autosomal recessive disease cystic fibrosis, modulates gut permeability, mucus production, and epithelial interactions with bacteria. The cystic fibrosis ΔF508 mutation is commonly found in the general population and has been shown to result in a reduced number of CFTR molecules at the surface of epithelial cells. Given the important biological functions of CFTR in the intestine, we tested whether this mutation is of relevance to IBD. Methods: Using DNA heteroduplex analysis, we investigated the distribution of ΔF508 heterozygosity in 2568 subjects from three independent cohorts of Italian, Swedish, and Scottish IBD patients and controls. Results: In all three cohorts an association between ΔF508 and Crohn's disease (CD) was observed. Specifically, ΔF508 heterozygosity was markedly underrepresented in CD patients from Italy and Sweden (P = 0.021 and 0.027 versus controls, respectively), while stratification for disease location revealed an absence of ΔF508 carriers among Scottish CD patients with right-sided colitis (P = 0.023 versus all other locations). Conclusions: ΔF508 heterozygosity might exert a protective effect in CD. (Inflamm Bowel Dis 2007)

Introduction Epigenetic alterations including DNA methylation may provide important insights into gene-environment interaction in complex immune diseases such as inflammatory bowel disease (IBD). Whilst whole tissue methylation changes may provide clinically useful biomarkers, epigenetic changes are cell-type specific. This study aimed to characterise the circulating methylome in IBD, and relate changes seen in whole blood to the methylation profile in separated leucocytes, gene expression data, as well as our previous data in childhood-onset disease. 1 Method The Illumina 450k array was used to assess whole blood leucocyte DNA methylation at over 485,000 CpG sites across the genome in 240 patients (121 Crohn’s disease [CD], 119 ulcerative colitis [UC]) and 191 controls. Differentially methylated sites discovered in whole blood were investigated in immunomagnetically separated leucocytes (CD4 + and CD8 + lymphocytes, CD14 + monocytes). Results There were 439 differentially methylated positions (DMPs) meeting epigenome wide significance as defined as a Holm corrected p value of –7 ) in IBD cases versus control. No markers were significantly different between CD and UC. There were 55 differentially methylated regions (DMRs) with unidirectional methylation change in 3 or more adjacent markers each achieving False Discovery Rate significance of p There was significant enrichment of methylation alteration around known susceptibility loci. 2 Linear Discriminant analysis using two CpG sites discriminated IBD cases and controls with high accuracy (area under curve 0.87). Established as well as novel pathways pertinent to disease pathogenesis are strongly implicated. The most significantly DMP in whole blood (RPS6KA2 [corrected p = 1.2 × 10 –16 ] was also hypomethylated in monocytes in UC (uncorrected p = 3.5 × 10 –6 ). The most significant DMR,VMP1/miR21 (most significant probe corrected p = 2 × 10 –14 ) strongly replicates the same finding in our previous study. The gene encoding Beta-2 Integrin (ITGB2) was a hypermethylated DMR in IBD and more specifically CD (most significant probe corrected p = 3.3 × 10 –5 ) compared with controls. Conclusion This is the most detailed characterisation of the epigenome carried out in IBD to date and includes novel data exploring the circulating methylome in UC. The findings strongly validate this approach in complex disease, replicate and expand previous data, and provide clear translational opportunities. Disclosure of interest N. Ventham Grant/Research Support from: EC grant IBD-BIOM, Conflict with: Abbvie, MSD speakers fees, N. Kennedy Grant/Research Support from: Wellcome Trust, Conflict with: Abbvie, MSD, Warner Chilcott, Ferring speakers fees. Shire Travel bursay, A. Adams: None Declared, R. Kalla: None Declared, K. O’Leary: None Declared, IBD BIOM consortium: None Declared, D. Wilson Grant/ Research Support from: MRC, CICRA, and EC grant IBD-BIOM, Consultant for: Pfizer, Conflict with: MSD investigator grant, MSD speaker fee, and Ferring speaker fee, E. Nimmo Grant/ Research Support from: EC grant IBD-BIOM, CSO, J. Satsangi Grant/ Research Support from: EC grant IBD-BIOM, Wellcome, CSO, MRC, Consultant for: Takeda, Conflict with: MSD speaker fees. SHire travelling expenses. References Adams AT, et al . IBD 2014;20(10):1784–93 Jostins L, et al . Nature 2012; 491(7422):119–24

Background: Although endoscopy is the gold standard for the assessment of activity in Crohn's disease (CD), it should be integrated with clinical, laboratory, and radiological data. In particular, it is still unclear when MRI should be performed. Aim: To evaluate the advantages and limitations of endoscopy and MRI to assess disease activity and severity in a series of CD patients. Material and methods: 50 consecutive patients with endoscopically provenCD underwentMRI enterography for the staging at diagnosis or follow-up. Endoscopic activity was measured by SES-CD (range 0-40) with active mild,moderate ad severe disease defined as a scores 4-10,11-19 and >20 respectively. MRI activity was measured by a validated quantitative score which integrates both mural and extramural involvement (Magnetic Resonance Enterography global score,MEGS,range 0-296), with active disease present for a ≥ 1 score. CDAI, CRP and fecal calprotectin (FC) were measured in all participants (positivity cut-off respectively >0,50 mg/dl and >150 μg/gr). Results: We enrolled 20 males and 30 females (diagnosis in 62%, follow-up in 38%), mean age 38±15 years,mean disease duration 5,4±5 years. SES-CD and MEGS were well correlated (r=0,42, p=0.001); both SESCD andMEGS show correlation with clinical (CDAI r=0,51,r=0,59, p

Crohn's disease (CD) is a chronic inflammatory bowel disorder caused by environmental and genetic factors. Mutations in the CARD15 gene have been associated with CD. No previous case-control CARD15 study has been performed in the Swedish population.The study comprised of 321 individuals: 178 with CD and 143 healthy controls (HCs), all from Stockholm County. All were genotyped for the three main CD-associated CARD15 variants (R702W, G908R and 1007fs) and phenotypic associations were investigated.The allele frequencies of the R702W variant (4.5% CD versus 0.7% HC, p=0.008, OR = 6.8) and the G908R variant (2.0% CD versus 0% HC, p=0.045) were more common in CD patients than in controls. No significant difference in1007fs variant allele frequency was found between CD patients and controls (2.0% CD versus 1.7% HC, p = 0.8, OR = 1.1). Carriage of CARD15 variants was more common in the CD patients than in controls (15.2% CD versus 4.2% HC, p = 0.001, OR = 4.1, population attributable risk (PAR) = 11.4%). Genotype-phenotype analysis demonstrated that CARD15 variants were associated with ileal disease (p=0.0006, OR = 9.3, CI = 2.2-34) and protective for colonic CD (p = 0.01, OR = 0.18). An association between CARD15 variants and ileal CD (p=0.004, OR = 6.6) was confirmed by multivariate analyses.The CARD15 variants R702W and G908R, but not 1007fs, are associated with susceptibility to CD in Stockholm County. Genotype-phenotype analysis shows an association with ileal CD. The contribution of these CARD15 mutations in Swedish CD patients overall is low in relation to studies elsewhere in Central Europe and North America, but is consistent with emerging data from elsewhere in Scandinavia and in Northern Europe.

Background: Crohn's disease (CD) is characterized by heterogeneity of phenotype. The Vienna classification can be used to classify CD, and recent data illustrate that behavior evolves over the course of the disease. Clinical and biological influences on disease progression remain unclear. We examined the associations of CD disease progression at diagnosis and for up to 20 years of follow-up. Methods: Two hundred thirty-one well-characterized CD patients were studied. Demographic, clinical, and NOD2/CARD15 data were collected. Disease behavior according to the Vienna classification was assessed at diagnosis and for up to 20 years following diagnosis. Results: At diagnosis, 70% of patients had inflammatory disease, 9% stricturing, and 21% penetrating. Early age at diagnosis was associated with ileocolonic and upper GI disease (p = 0.015), and positive anti-Saccharomyces cerevisiae antibody (ASCA) was associated with ileal invovement (p = 0.008). Smoking was relatively protective against colonic, rather than ileal involvement at diagnosis (p < 0.02). At 20 years, 92% had progressed to a more severe disease type. Patients who progress to a more severe disease type require more frequent surgery (p < 0.00001). Multivariate analysis found disease progression to be associated with ileal disease location (p = 0.001) and positive ASCA (p = 0.003). Variant NOD2/CARD15 alleles were protective against rapid progression of disease phenotype (p = 0.04). The presence of perianal disease was independent of intestinal penetrating disease. Conclusions: The progression of disease type in CD is associated with the need for more frequent surgery. Rapid progression is associated with ileal disease and positive ASCA, and delayed progression is associated with variant NOD2/CARD15 alleles. Consideration should be given to a separate Vienna classification for perianal disease.

NOD2/caspase recruitment domain (CARD)15 variants are identified in up to 50% of Crohn's disease (CD) patients. Functional variants of toll-like receptor-4 (TLR4) and CD14 genes may also be relevant to disease pathophysiology. We aimed to assess the contribution of NOD2/CARD15, TLR4 and CD14 variants in Scottish and Irish CD patients. In all, 612 patients with well-characterised inflammatory bowel disease (252 Scottish CD, 247 Scottish UC, 113 Irish CD) and 304 controls were genotyped for variants of NOD2/CARD15 (1007fsinsC, G908R, R702W, P268S), TLR4 (A299G) and CD14 (T-159C). Genotype–phenotype analyses were performed. Variant 1007fsinsC (P=0.003) and G908R (P=0.008) but not R702W (P=0.269) alleles were more prevalent in Scottish CD (4.7, 1.8 and 7.1%, respectively) than Scottish control (2.3, 0.3 and 5.4%). CD allelic frequencies were lower than the series from Europe (P

Introduction DNA methylation influences transcriptional activity and marks sites of active transcription. Technological developments allow the rapid assessment of methylation state at 450,000 sites across the genome. The aim of this study was to identify genes with a possible role in Crohn’s disease pathogenesis, and candidate genes for methylation based diagnostic biomarkers. Methods Using the Illumina 450k platform we analysed genome-wide DNA methylation in symptomatic children who underwent diagnostic colonoscopy, half of whom were diagnosed with CD and half had no pathology. Replication was performed in children with established CD vs. symptomatic non-disease controls. Further targeted replication by pyrosequencing was performed in adults with CD vs. healthy controls, with qPCR and microarray data to analyse expression. Results Meta-analysis of the combined paediatric datasets (n = 66) identified 165 individual CpGs with epigenome-wide significance (Bonferroni correction) and 138 differentially methylated regions (DMR). Methylation changes were significantly enriched (p The strongest result by each approach was MicroRNA 21, within the autophagy gene VMP1 (p = 1.2 × 10 –14 ), 48 kb from GWAS SNP rs1292053. In adults with CD we replicated MIR21 hypomethylation (p = 6.6 × 10 –5 , n = 172), and showed increased expression in blood (p –6 , n = 99) but not controls (n = 73). Linear discriminant analysis of methylation in the paediatric discovery cohort accurately predicted disease state in the paediatric replication cohort (94% sensitivity, 100% specificity) based on methylation at two CpG sites. Conclusion MIR21 emerges as a target for further investigation based on methylation and expression, further strengthened by other positive findings – notably dysregulation in dysplasia and colorectal cancer in IBD, an established role in T-cell differentiation, and protection from DSS-induced fatal colitis by MIR21 knockout. These data demonstrate a novel approach for identifying biological variations associated with germ-line variants identified by GWAS, and demonstrate translational potential for biomarker development and therapeutic target discovery. Disclosure of Interest None Declared.

in the CD4+aEb7 compartment (p < 0.001). IL-10 was not differentially expressed between CD4+aEb7+ and CD4+aEb7 lymphocytes in controls or UC, and a low frequency of aEb7+FOXP3+ cells was observed by IHC. qPCR array confirmed higher mRNA levels of IFNg (p < 0.001), TNFa (p < 0.01) and IL-17A (p < 0.01), and lower transcription of FOXP3 (p < 0.0001) in CD4+aEb7+ cells compared to CD4+aEb7 cells. Conclusions: aEb7 expression was associated with an enrichment of pro-inflammatory Th17, Th1 and Th17/Th1 T lymphocytes, and not associated with a regulatory phenotype. These data suggest therapeutic interventions targeting aE expressing T cells and the aEb7 integrin itself may be viable approaches for reducing aberrant inflammatory responses in UC.

To the Editor:Since the discovery of NOD2 as aCrohn’s disease (CD) susceptibilitygene, more recent genomewide associa-tion scans (GWAS), detecting loci con-ferring odds ratios (OR) of above 1.3–1.5, have uncovered a large number offurther susceptibility loci and genes,including IL23R in the TH17 pathway,and the autophagy genes ATG16L1 andIRGM.

Genetic association of the autophagy genes, autophagy-related 16-like 1 (ATG16L1) and immunity-related GTPase family, M (IRGM), has been confirmed by meta-analysis of three index genome-wide association studies (GWASs), and in other independent studies in adult-onset Crohn’s disease, but not ulcerative colitis.1–5 While the meta-analysis implicated a single nucleotide polymorphism (SNP; rs11747270) based on imputed data from the constituent GWAS with odds ratio (OR) 1.33, other studies have replicated the IRGM signal based on rs13361189 and rs4958847. Recently, rs13361189, upstream of IRGM, was shown to be in perfect linkage disequilibrium with a 20 kb deletion polymorphism affecting the expression of IRGM (and cellular autophagy) in a tissue-specific manner.5 We read with interest the letter by Peterson et al ( Gut 2008; 57 :1336–7) which suggested association of IRGM with childhood-onset Crohn’s disease in a North American cohort. Only one of the two IRGM variants studied (rs13361189) was weakly associated with Crohn’s disease in their study …

The sequence requirements for in vivo telomere function in the fission yeast, Schizosaccharomyces pombe, have been investigated. A 258 bp tract of previously characterized cloned fission yeast terminal repeats adjacent to 800 bp of telomere-associated sequences is sufficient to seed new telomeres onto linearized ars-containing plasmids when introduced into cells. The resulting transformants contain unrearranged, acentric, linear episomes. Cloned telomeres, with and without telomere-associated sequences adjacent to the 258 bp terminal repeats, were utilized to introduce chromosome breaks at specific sites in a non-essential minichromosome. Truncated minichromosome derivatives were recovered containing the ura4 or ade6 gene adjacent to a newly formed telomere. These telomeres exert reversible position effects on the expression of the adjacent ura4 or ade6 genes.

Background & Aims The mechanisms by which specific mutations in NOD2 / CARD15 increase the risk for Crohn's disease (CD) are unclear. We identified proteins that interact with NOD2 and investigated them by expression, genetic, and functional analyses. Methods By using a yeast 2-hybrid screen of an intestinal epithelial library, we identified proteins that interact with NOD2 and confirmed the interactions in mammalian cells using co-immunoprecipitation. We used microarray analysis to analyze gene expression patterns in 302 intestinal biopsy samples (129 from patients with ulcerative colitis [UC], 106 with CD, and 67 controls). Eighty single-nucleotide polymorphisms within the genes that encoded 6 interacting proteins were genotyped in a discovery cohort (869 cases of inflammatory bowel disease [IBD], 885 controls) and a replication cohort (504 patients with IBD, 713 controls). We investigated interaction between transducin-like enhancer of split 1 (TLE1) and NOD2 in HEK293 cells. Results We identified 6 NOD2-interacting proteins (TLE1, UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2 [GALNT2], HIV-1 Tat interactive protein [HTATIP], Vimentin, fission 1 (mitochondrial outer membrane) homolog [FIS1], and protein phosphatase 2, regulatory subunit B′, epsilon isoform [PPP2R5E]). Of these, expression of GALNT2 (CD, P = .004) and vimentin (CD, P = .006; UC, P = .0025) was altered in patients with IBD compared with controls. Single-nucleotide polymorphisms within TLE1 were associated with susceptibility to CD, specifically with ileal disease (rs6559629, P = 3.1 × 10 −5 ; odds ratio, 1.45). The TLE1 risk allele is required for susceptibility to CD in carriers of NOD2 mutations. In cells, TLE1 and NOD2 co-localized around the nuclear membrane and TLE1 inhibited activation of nuclear factor-κB by NOD2. Conclusions Epistatic and biological interactions between TLE1 and NOD2 are involved in IBD pathogenesis. NOD2 might be involved in a series of pathways such as epigenetic regulation of expression (via TLE1 and HTATIP), biosynthesis of mucin (via GALNT2), apoptosis (via PPP2R5E and FIS1), and integrity of the intracellular cytoskeleton (vimentin).